It has been known for several years that certain short nucleic acid sequences are able to demonstrate a significant physiological effect, by stimulating effector cells in the immune system via unknown mechanisms. These short nucleic acid sequences, which are generally referred to as immunostimulatory nucleic acid sequences (ISSs), are only a few bases in length, and their functions are usually not dependent upon expression of proteins encoded by them.
Most known immunomodifying cytosine oligodeoxyribo-nucleotide sequences (ODNs) contain at least one CpG motif. Krieg et al., CpG Motifs in Bacterial DNA Trigger Direct B-Cell Activation, Nature 374:6522 546-9 (Apr. 6, 1995). The occurrence of CpG motifs in the genome of eukaryotes is substantially less than that in the genome of prokaryotes. It is therefore suggested that recognition of CpG motifs by eukaryotic cells may be used as a warning signal to indicate infection by prokaryotic pathogens. For instance, recognition of the CpG motifs by the eukaryotic cells may lead to certain emergency responses in the cells, which then trigger a reaction directed against the viral or bacterial pathogens, independently of or prior to the production of a T-helper cell immmunoresponse. In fact, CpG motifs trigger certain CpG-dependent signal paths, which generates costimulatory signals that are required for activating T-cells and B-cells, in particular for the secretion of cytokines by a cellular Type-1 response (Th1-specific cytokines such as interferon gamma, IL-7, IL-12), thus enabling stimulation and proliferation of B-cells independent of T-helper cells. Moreover, the activities of the Type-2 cytokines (such as IL-4 and IL-10) are suppressed by such CpG-dependent signal paths, probably because of the antagonism effect between Type-1 and Type-2 responses. Sedar & Paul, Acquisition of Lumphokine-Producing Phenotype by CD4+ T Cells, Annual Rev. Immunol., 12:635-73 (1994).
The potential of using nucleic acid molecules containing CpG motifs to modulate the immunoresponse is considerable, which has generated sudden and widespread scientific interest in exploiting the therapeutic and prophylactic applications of such molecules.
It has been discovered that CpG motifs are more effective as immunostimulators when they exist as single-stranded molecules. See WO98/18810 A1, S. 17, II 29-30. Therefore, numerous experimental approaches aiming to treat infectious illnesses, tumors, and/or autoimmune diseases use short, open-chain, single-stranded ISS oligodeoxynucleotides that contain CpG motifs.
However, the open-chain, single-stranded ISS oligodeoxynucleotides degrade very quickly in vivo, due to impacts by extracellular and intracellular exonucleases. Moreover, the active single-stranded ISS molecules, due to their instability, are too toxic for direct use in human medical applications. Therefore, the use of isolated ISS oligodeoxynucleotides in in vivo applications is not practicable. They have to be either modified before in vivo administration or introduced into vector sequences. WO98/18810 A1; Weeratna et al., Reduction of Antigen Expression from DNA Vaccines by Coadministered Oligodeoxynucleotides, Antisense Nucleic Acid Drug Dev., 8(4):351-6 (August 1998).
Extracellular and intracellular exonucleases have been found to display significantly reduced enzymatic activity when modified phosphor-ester bonds are formed in the backbone of the open-chain, single-stranded ISS oligodeoxynucleotides. This discovery has led to use of phosphor thioesters (“thioates”) or reduced phosphor bonds (phosphonates) in chiral or achiral form to stablize the open-chain, single-stranded nucleic acid molecules that are to be administered to patients.
These modified bonds can be produced by the solid phase synthesis method. However, such method is much more complicated than the classic DNA amidites synthesis method. Moreover, during clinical studies of antisense strategies, where these modified bonds are frequently used, it was discovered, that these modified bonds cause considerable amount of side effects, particularly on the blood coagulation system and the complementary system. Sheehan & Lan, Blood 92, 1617-25 (1998). Furthermore, when these modified bonds are introduced into the ISS molecules to form thiophosphoric acid derivatives for the purpose of stabilizing the ISS molecules, the resulted ISS molecules display less stimulatory activities, due to the fact that the CpG motifs, which are required for the stimulatory activities to be effectuated, are themselves protected by the flanking sequences. WO98/18810 A1.
WO98/18810 A1 comprehensively describes the theories concerning use and production of immunostimulatory ISS molecules containing CpG motifs. It also presents several solutions to the problem of in vivo instability of such molecules, including formation of thiophosphate esters, dithiophosphate esters or phosphonates and creation of secondary structures (such as a stem-loop). However, these solutions presented and suggested by WO98/18810 are expressly limited to single-stranded linear ODNs.
U.S. Pat. Nos. 5,663,153, 5,723,335, and 5,856,462 disclose production and use of phosphorothioate oligomers in connection with ISS molecules.
U.S. Pat. No. 5,750,669 suggests a different approach for protecting open-chain, single-stranded ISS molecules, which relates to linking the ends of the oligomers with nucleoside residues via 5′-5′ and 3′-3′ bonds, which functions to block exonucleolytic degradation of the ISS molecules.
Hoson et al., Biochim. Biophys. Acta. 244, 339-344 (1995), disclose formation of linear oligodeoxynucleotides with a stem-loop structure at the 3′ end, which can be used for antisense research.
Double stem-loop or covalently closed, dumbbell-shaped ODNs are known from experimental approaches that focus on competition in bonding sites for DNA-binding proteins and transcription factors. Lim et al., Nuc. Acids Res. 25, 575-581 (1997); Blumenfeld et al., Nuc. Acids. Res. 21, 3405-3411 (1993).